Multivalent pneumococcal polysaccharide-protein conjugate composition

ABSTRACT

Provided are mixed carrier, multivalent pneumococcal conjugate compositions comprising 16 different pneumococcal capsular polysaccharide-protein conjugates, wherein each of the conjugates includes a capsular polysaccharide from a different serotype of  Streptococcus pneumoniae  conjugated to either tetanus toxoid or CRM 197 , wherein the  Streptococcus pneumoniae  serotypes are selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and 33F, wherein two of the capsular polysaccharides are conjugated to tetanus toxoid and the remaining capsular polysaccharides are conjugated to CRM 197 , and wherein the two capsular polysaccharides that are conjugated to tetanus toxoid are selected from the group consisting of serotypes 1, 3, and 5. Also provided are methods of producing the mixed carrier, multivalent pneumococcal conjugate compositions and methods of using the same for prophylaxis against  Streptococcus pneumoniae  infection or disease in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and relies on the filing dateof, U.S. provisional patent application number 62/371,529. filed 5 Aug.2016, the entire disclosure of which is herein incorporated byreference.

TECHNICAL FIELD

This application relates generally to mixed carrier, multivalentpneumococcal conjugate compositions, vaccines comprising the same andmethods of using these compositions and vaccines for prophylaxis ofStreptococcus pneumoniae infection or disease in a subject.

BACKGROUND

Pneumococcus (Streptococcus pneumoniae) is a Gram-positive,lancet-shaped, facultative anaerobic bacteria with over 90 knownserotypes Most S. pneumoniae serotypes have been shown to cause disease,with the 23 most common serotypes accounting for approximately 90% ofinvasive disease worldwide. Serotypes are classified based on theserological response of the capsular polysaccharides, the most importantvirulence factor for pneumococcus. Capsular polysaccharides are T-cellindependent antigens that induce antibody production in the absence of Thelper cells. T-cell independent antigens generally induce antibodieswith low affinity and short-lived immune responses with little to noimmunological memory.

Initial pneumococcal vaccines included combinations of capsularpolysaccharides from different serotypes. These vaccines can conferimmunity against S. pneumoniae in patients with developed or healthyimmune systems, however, they were not effective in infants, who lack adeveloped immune system, and elderly subjects, who often have impairedimmune function. To improve the immune response to pneumococcalvaccines, particularly in infants and elderly subjects, who are athigher risk to develop S. pneumoniae infection, capsular polysaccharideswere conjugated to suitable carrier proteins to create pneumococcalconjugate vaccines. Conjugation to a suitable carrier protein changesthe capsular polysaccharide from a T-cell independent antigen to aT-cell dependent antigen. As such, the immune response against theconjugated capsular polysaccharide involves T helper cells, which helpinduce a more potent and rapid immune response upon re-exposure to thecapsular polysaccharide.

There are at least two approaches to developing pneumococcal conjugatevaccines: the single carrier approach and the mixed carrier approach.The immunogenicity of different capsular polysaccharide conjugates mayvary depending on the pneumococcal serotype and carrier protein used. Inthe single carrier approach, the capsular polysaccharides from differentserotypes are conjugated to a single protein carrier. Pfizer's PREVNARseries of vaccines is an example of a single carrier approach where thedifferent capsular polysaccharides are conjugated to the CRM₁₉₇ proteincarrier, a non-toxic variant of the diphtheria toxoid having a singleamino acid substitution of glutamic acid for glycine. The 7-valentPREVNAR vaccine (PREVNAR) was first approved in 2000 and contains thecapsular polysaccharides from the seven most prevalent serotypes: 4, 6B,9V, 14, 18C, 19F and 23F. The 13-valent vaccine, PREVNAR 13, added theserotypes 1, 5, 7F, 3, 6A, and 19A to the CRM₁₉₇ protein carrier. Theprotein carrier, CRM₁₉₇, used in the single carrier, PREVNAR vaccineshas never been used as part of a mixed carrier system in a pneumococcalconjugate vaccine.

The second pneumococcal vaccine approach is the mixed carrier approach.In the mixed carrier approach, instead of using a single proteincarrier, two or more protein carriers are used, with capsularpolysaccharides from specific serotypes conjugated to a first proteincarrier and capsular polysaccharides from different serotypes conjugatedto at least a second, different protein carrier. For example,GlaxoSmithKline has developed SYNFLORIX, a 10-valent (serotypes 1, 4, 5,6B, 7F, 9V, 14, 18C, 19F and 23F), mixed carrier, pneumococcal conjugatevaccine that uses H influenzae protein D, tetanus toxoid, and diphtheriatoxoid as the protein carriers. In SYNFLORIX, serotypes 1, 4, 5, 6B, 7F,9V, 14, and 23F are conjugated to protein serotype 18C is conjugated totetanus toxoid; and serotype 19F is conjugated to diphtheria toxoid [7].Serotype 3 was removed from the 11-valent precursor to SYNFLORIX becauseit did not show serotype-specific efficacy in an acute otitis mediatrial [1]. Another group, Aventis Pasteur, developed an 11-valent(serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F), mixedcarrier, pneumococcal conjugate vaccine using diphtheria toxoid andtetanus toxoid as protein carriers [2, 3]. Capsular polysaccharides fromserotypes 3, 9V, 14, and 18C can evoke a better response when conjugatedto diphtheria toxoid than they do when conjugated to tetanus toxoid [2,6]. Thus, serotypes 3, 6B, 14, and 18C were conjugated to diphtheriatoxin and serotypes 1, 4, 5, 7F, 9V, 19F, and 23F were conjugated totetanus toxoid. The development of this mixed carrier, pneumococcalvaccine was terminated due, in part, to technical reasons and thepotential of a reduced response when combined with acellular pertussisvaccines [3]. Recently, serotype 5 as well as 1 was reported as havingone of the lowest observed OPA titres from all PREVNAR 13 serotypes, inwhich there was an associated correlation between IgG titer and OPAactivity [4]. Also it was suggested that for serotype 3, a much higherserum IgG concentration would be needed for protection [5].

SUMMARY

This application provides new and improved mixed carrier, multivalentpneumococcal conjugate compositions and vaccines comprising the same. Inone aspect, this application provides a mixed carrier, multivalentpneumococcal conjugate composition, comprising 16 different pneumococcalcapsular polysaccharide-protein conjugates, wherein each pneumococcalcapsular polysaccharide-protein conjugate comprises a protein carrierconjugated to a capsular polysaccharide from a different serotype ofStreptococcus pneumoniae, wherein the Streptococcus pneumoniae serotypesare selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F,22F, 23F, and 33F, wherein the protein carrier is CRM₁₉₇ or tetanustoxoid, wherein two of the capsular polysaccharides are conjugated totetanus toxoid and the remaining capsular polysaccharides are conjugatedto CRM₁₉₇, and wherein the two capsular polysaccharides that areconjugated to tetanus toxoid are selected from the group consisting ofserotypes 1, 3, and 5.

In some embodiments, the capsular polysaccharides from serotypes 1 and 5are conjugated to tetanus toxoid, and the capsular polysaccharides fromserotypes 3, 4, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and33F are conjugated to CRM₁₉₇.

In another embodiment, the capsular polysaccharides from serotypes 1 and3 are conjugated to tetanus toxoid, and the capsular polysaccharidesfrom serotypes 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F,and 33F are conjugated to CRM₁₉₇.

In yet another embodiment, the capsular polysaccharides from serotypes 3and 5 are conjugated to tetanus toxoid, and the capsular polysaccharidesfrom serotypes 1, 4, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F,and 33F are conjugated to CRM₁₉₇.

In some embodiments, the mixed carrier, multivalent pneumococcalconjugate composition further comprises an adjuvant, such as analuminum-based adjuvant, including, but not limited to aluminumphosphate, aluminum sulfate, and aluminum hydroxide.

Another aspect is directed to the use of the mixed carrier, 16-valentpneumococcal conjugate composition as a vaccine.

Yet another aspect is directed to a vaccine comprising the mixedcarrier, 16-valent pneumococcal conjugate composition and apharmaceutically acceptable excipient.

Yet another aspect is directed to a method for prophylaxis ofStreptococcus pneumoniae infection or disease in a subject, such as ahuman, the method comprising administering a prophylactically effectiveamount of the mixed carrier, 16-valent pneumococcal conjugatecompositions or a vaccine comprising the same to the subject.

In certain embodiments, the subject is a human who is at least 50 yearsold and the disease is pneumonia or invasive pneumococcal disease (IPD).

In other embodiments, the subject is a human who is at least 6 weeks oldand the disease is pneumonia, invasive pneumococcal disease (IPD), oracute otitis media (AOM). In some embodiments, the human subject is 6weeks to 5 years of age. In other embodiments, the human subject is 2 to15 months of age or 6 to 17 years of age.

In certain embodiments, the mixed carrier, 16-valent pneumococcalconjugate composition or vaccine is administered by intramuscularinjection. In certain embodiments, the mixed carrier, 16-valentpneumococcal conjugate composition or vaccine is administered as part ofan immunization series.

The foregoing and other objects, features, and advantages of the mixedcarrier, 16-valent pneumococcal conjugate compositions will become moreapparent from the following detailed description.

Definitions

In order for the present disclosure to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms may be set forth through thespecification.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Adjuvant: As used herein, an “adjuvant” refers to a substance or vehiclethat non-specifically enhances the immune response to an antigen.

Administer: As used herein, “administering” a composition to a subjectmeans to give, apply or bring the composition into contact with thesubject. Administration can be accomplished by any of a number ofroutes, such as, for example, topical, oral, subcutaneous,intramuscular, intraperitoneal, intravenous, intrathecal andintradermal.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Conjugate: As used herein, and understood from the proper context, theterms “conjugate(s)” or “glycoconjugate(s)” refer to a Streptococcuspneumoniae polysaccharide conjugated to a carrier protein using anycovalent or non-covalent bioconjugation strategy.

Excipient: As used herein, the term “excipient” refers to anon-therapeutic agent that may be included in a composition, for exampleto provide or contribute to a desired consistency or stabilizing effect.

Mixed carrier. As used herein, a mixed carrier, pneumococcal conjugatecomposition refers to a pneumococcal conjugate composition having morethan one type of protein carrier.

Multivalent: As used herein, the term “multivalent” refers to apneumococcal conjugate composition having pneumococcal capsularpolysaccharides from more than one Streptococcus pneumoniae serotype.

Mixed carrier, 16-valent pneumococcal conjugate composition: As usedherein, the term “mixed carrier, 16-valent pneumococcal conjugatecomposition(s)” refers to a composition comprising 16 differentpneumococcal capsular polysaccharide-protein conjugates, wherein eachpneumococcal capsular polysaccharide-protein conjugate comprises aprotein carrier conjugated to a capsular polysaccharide from a differentserotype of Streptococcus pneumoniae, wherein the Streptococcuspneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A,19F, 22F, 23F, and 33F, wherein the protein carrier is CRM₁₉₇ or tetanustoxoid, wherein two of the capsular polysaccharides are conjugated totetanus toxoid and the remaining capsular polysaccharides are conjugatedto CRM₁₉₇, and wherein the two capsular polysaccharides that areconjugated to tetanus toxoid are selected from the group consisting ofserotypes 1, 3, and 5. In some embodiments, the capsular polysaccharidesfrom serotypes 1 and 5 are conjugated to tetanus toxoid, and thecapsular polysaccharides from the remaining serotypes are conjugated toCRM₁₉₇. In another embodiment, the capsular polysaccharides fromserotypes 1 and 3 are conjugated to tetanus toxoid, and the remainingcapsular polysaccharides are conjugated to CRM₁₉₇. In yet anotherembodiment, the capsular polysaccharides from serotypes 3 and 5 areconjugated to tetanus toxoid, and the remaining capsular polysaccharidesare conjugated to CRM₁₉₇.

Pharmaceutically acceptable excipient: The pharmaceutically acceptableexcipients useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of one or more therapeuticcompositions, including vaccines, and additional pharmaceutical agents.Suitable pharmaceutical excipients include, for example, starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. In general, the nature of the excipient will depend on theparticular mode of administration being employed. For instance,parenteral formulations usually comprise injectable fluids that includepharmaceutically and physiologically acceptable fluids such as water,physiological saline, balanced salt solutions, buffers, aqueousdextrose, glycerol or the like as a vehicle. For solid compositions (forexample, powder, pill, tablet, or capsule forms), conventional non-toxicsolid excipients can include, for example, pharmaceutical grades ofmannitol, lactose, starch, or magnesium stearate. In addition tobiologically-neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, a surface activeagent, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Prophylactically effective amount: As defined herein, the term “aprophylactically effective amount” or “a prophylactically effectivedose” refers to the amount or dose required to induce an immune responsesufficient to delay onset and/or reduce in frequency and/or severity oneor more symptoms caused by an infection with Streptococcus pneumoniae.

Prophylaxis: The term “prophylaxis,” as used herein, refers to avoidanceof disease manifestation, a delay of onset, and/or reduction infrequency and/or severity of one or more symptoms of a particulardisease, disorder or condition (e.g., infection with Streptococcuspneumoniae). In some embodiments, prophylaxis is assessed on apopulation basis such that an agent is considered to provide prophylaxisagainst a particular disease, disorder or condition if a statisticallysignificant decrease in the development, frequency, and/or intensity ofone or more symptoms of the disease, disorder or condition is observedin a population susceptible to the disease, disorder, or condition.

Subject: As used herein, the term “subject” means any mammal, includingmice, rabbits, and humans. In certain embodiments, the subject is anadult, an adolescent or an infant. In some embodiments, terms“individual” or “patient” are used and are intended to beinterchangeable with “subject.”

DETAILED DESCRIPTION

The following description of the disclosed embodiment(s) and Examples ismerely exemplary in nature and is in no way intended to limit theinvention, its application, or uses.

This application provides new and improved mixed carrier, multivalentpneumococcal conjugate compositions and vaccines comprising the same.While the protein carrier, CRM₁₉₇, has previously been used in singlecarrier, pneumococcal conjugate vaccines, this application describes forthe first time the use of CRM₁₉₇ in a mixed carrier, pneumococcalconjugate vaccine.

As discussed above, the immunogenicity of different capsularpolysaccharide conjugates may vary depending on the pneumococcalserotype and carrier protein used. This application describes thesuccessful conjugation of serotype 3 to tetanus toxoid as part of amixed carrier vaccine, notwithstanding previous teachings that serotype3 was more immunogenic when conjugated to diphtheria toxoid rather thantetanus toxoid [2, 6]. It also discloses the unexpected finding that theantibody response to serotype 3 conjugated to tetanus toxoid in a mixedcarrier, 16-valent pneumococcal conjugate composition was about 4-foldhigher than when serotype 3 was conjugated to CRM₁₉₇ in a singlecarrier, 13-valent pneumococcal conjugate composition (PREVNAR 13).

Further, the unexpected finding was not limited to serotype 3 but wasalso observed for the other serotypes conjugated to tetanus toxoid inthe mixed carrier, 16-valent pneumococcal conjugate composition. Asshown in the Examples, conjugation of serotypes 1 and 3, 1 and 5, or 3and 5 to tetanus toxoid in a mixed carrier, pneumococcal conjugatecomposition with the remaining serotypes conjugated to CRM₁₉₇(PVC16-13TT, PVC16-15TT, and PVC16-35TT, respectively) consistentlyinduced significantly enhanced antibody responses to the serotypesconjugated to tetanus toxoid as compared to the antibody responses (IgGresponse or MOPA titers) against the same serotypes conjugated to CRM₁₉₇in a single carrier, pneumococcal conjugate composition (PREVNAR 13), assummarized in the table below.

TABLE 1 Fold Increase in Antibody Response to Serotypes Conjugated toTetanus Toxoid in Mixed Carrier Vaccine Compared to PREVNAR 13 FoldIncrease in Antibody Response Compared to PREVNAR 13 Serotype PVC16-13TTPVC16-15TT PVC16-35TT 1 (IgG)  3X 3.9X N/A 1 (MOPA) 6.3X 6.3X N/A 3(IgG) 5.5X N/A 2.8X 3 (MOPA)  4X N/A  4X 5 (IgG) N/A 8.7X 3.2X 5 (MOPA)N/A  5X  4X

The mixed carrier, 16-valent pneumococcal conjugate compositionsdescribed in this application also include pneumococcal serotypes notcovered by the three pneumococcal conjugate vaccines currently availableon the global market: PREVNAR (called Prevenar in some countries),SYNFLORIX and PREVNAR 13. Disease caused by pneumococcal serotypes notcurrently covered is on the rise, due, in part, to the development ofantibacterial resistance, the increased number of immunocompromisedpatients, and lack of immune pressure. For example, none of thecurrently available pneumococcal conjugate vaccines includes serotype12F. In addition, none of the currently available pneumococcal conjugatevaccines includes serotypes 22F and 33F. The present disclosuredemonstrates the successful implementation of serotypes 12F, 22F, and33F into a mixed carrier, pneumococcal conjugate vaccine.

Mixed Carrier, Multivalent Pneumococcal Conjugate Compositions andMethods of Making the Same

In one aspect, this disclosure provides a mixed carrier, multivalentpneumococcal conjugate composition comprising 16 different pneumococcalcapsular polysaccharide-protein conjugates, wherein each pneumococcalcapsular polysaccharide-protein conjugate comprises a protein carrierconjugated to a capsular polysaccharide from a different serotype ofStreptococcus pneumoniae, wherein the Streptococcus pneumoniae serotypesare 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and33F, wherein the protein carrier is CRM₁₉₇ or tetanus toxoid, whereintwo of the capsular polysaccharides are conjugated to tetanus toxoid andthe remaining capsular polysaccharides are conjugated to CRM₁₉₇, andwherein the two capsular polysaccharides that are conjugated to tetanustoxoid are selected from the group consisting of serotypes 1, 3, and 5.

In some embodiments, the capsular polysaccharides from serotypes 1 and 5are conjugated to tetanus toxoid, and the capsular polysaccharides fromthe remaining serotypes are conjugated to CRM₁₉₇. In another embodiment,the capsular polysaccharides from serotypes 1 and 3 are conjugated totetanus toxoid, and the remaining capsular polysaccharides areconjugated to CRM₁₉₇. In yet another embodiment, the capsularpolysaccharides from serotypes 3 and 5 are conjugated to tetanus toxoid,and the remaining capsular polysaccharides are conjugated to CRM₁₉₇.

In a polysaccharide-protein conjugate vaccine, a carrier protein isconjugated to a polysaccharide antigen primarily to help enhance theimmune response (e.g. antibody response) to the polysaccharide antigen.Carrier proteins are preferably proteins that are non-toxic havinglittle or no immunogenicity. Carrier proteins should be amenable toconjugation with a pneumococcal polysaccharide using standardconjugation procedures, as discussed in further detail below. Thecarrier proteins used in the mixed carrier, 16-valent pneumococcalconjugate compositions are tetanus toxoid (TT) and CRM₁₉₇, each of whichhas been used in the design of pneumococcal conjugate vaccines but neverin the same, mixed carrier vaccine.

CRM₁₉₇ is a non-toxic variant (i.e., toxoid) of diphtheria toxin thatretains the immunologic properties of the wild type diphtheria toxin.CRM₁₉₇ differs from the wild type diphtheria toxin at a single base inthe structural gene, which gives rise to a single amino acidsubstitution from glutamic acid to glycine. CRM₁₉₇ is typically isolatedfrom cultures of Corynebacterium diphtheria strain C7 (β197) grown oncasamino acids and yeast extract-based medium. CRM₁₉₇ may be purifiedthrough ultra-filtration, ammonium sulfate precipitation, andion-exchange chromatography. Alternatively, CRM₁₉₇ can be preparedrecombinantly in accordance with U.S. Pat. No. 5,614,382, which ishereby incorporated by reference in its entirety. CRM₁₉₇ has been usedin the design of pneumococcal conjugate vaccines but never as part of amixed carrier vaccine.

Tetanus toxoid is prepared and used worldwide for large-scaleimmunization against tetanus (or lockjaw) caused by Clostridium tetani.Tetanus toxoid is also used both singly and in combination withdiphtheria and/or pertussis vaccines. The parent protein, tetanus toxin,is generally obtained in cultures of Clostridium tetani. Tetanus toxinis a protein of about 150 kDa and consists of two subunits (about 100kDa and about 50 kDa) linked by a sulfide bond. The toxin is typicallydetoxified with formaldehyde and can be purified from culture filtratesusing known methods, such as ammonium sulfate precipitation (see, e.g.,[7], [8]) or chromatography techniques, as disclosed, for example, in WO1996/025425. Tetanus toxin may also be inactivated by recombinantgenetic means.

Tetanus toxoid has also been used as a carrier protein in othervaccines, including pneumococcal conjugate vaccines. But it has neverbeen used in a mixed carrier, pneumococcal conjugate vaccine incombination with CRM₁₉₇ The art also teaches away from conjugatingserotype 3 to tetanus toxoid in a mixed carrier, pneumococcal conjugatevaccine because serotype 3 was shown to be more immunogenic whenconjugated to diphtheria toxoid as compared to tetanus toxoid [2, 6].

The pneumococcal capsular polysaccharides used in the compositions andvaccines described herein, including the capsular polysaccharides fromserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F,and 33F, may be prepared from Streptococcus pneumoniae using anyavailable technique, including standard techniques known to one ofordinary skill in the art, including, for example, those disclosed in WO2006/110381, WO 2008/118752, WO 2006/110352, and U.S. Patent App. Pub.Nos. 2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072,2007/0231340, 2008/0102498 and 2008/0286838, all of which areincorporated by reference in their entireties. For example, eachpneumococcal capsular polysaccharide serotype may be grown in culturemedium (e.g., a soy-based medium). The cells are lysed, and individualpolysaccharides may be purified from the lysate through centrifugation,precipitation, ultra-filtration, and/or column chromatography. Inaddition, the pneumococcal capsular polysaccharide can be produced usingsynthetic protocols.

Capsular polysaccharides of Streptococcus pneumoniae comprise repeatingoligosaccharide units, which may contain up to 8 sugar residues. Acapsular saccharide antigen may be a full length polysaccharide, or itmay be reduced in size (e.g., a single oligosaccharide unit, or ashorter than native length saccharide chain of repeating oligosaccharideunits). The size of capsular polysaccharides may be reduced by variousmethods known in the art, such as acid hydrolysis treatment, hydrogenperoxide treatment, sizing by a high pressure homogenizer, optionallyfollowed by a hydrogen peroxide treatment to generate oligosaccharidefragments, or microfluidization.

The pneumococcal conjugate of each of the serotypes may be prepared byconjugating a capsular polysaccharide of each serotype to a carrierprotein. The different pneumococcal conjugates may be formulated into acomposition, including a single dosage formulation.

To prepare a polysaccharide-protein conjugate, the capsularpolysaccharides prepared from each pneumococcal serotype may bechemically activated so that the capsular polysaccharides may react witha carrier protein. Once activated, each capsular polysaccharide may beseparately conjugated to a carrier protein to form a glycoconjugate. Thechemical activation of the polysaccharides and subsequent conjugation tothe carrier protein may be achieved by conventional methods. Forexample, vicinal hydroxyl groups at the end of the capsularpolysaccharides can be oxidized to aldehyde groups by oxidizing agentssuch as periodates (including sodium periodate, potassium periodate, orperiodic acid), as disclosed, for example, in U.S. Pat. Nos. 4,365,170,4,673,574 and 4,902,506, which are hereby incorporated by reference intheir entireties. Polysaccharides may also be activated with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. The activated polysaccharide is then coupled directly orvia a spacer or linker group to an amino group on the carrier protein.

For example, the spacer could be cystamine or cysteamine to give athiolated polysaccharide which could be coupled to the carrier via athioether linkage obtained after reaction with a maleimide-activatedcarrier protein (for example using N-[y-maleimidobutyrloxy]succinimideester (GMBS)) or a haloacetylated carrier protein (for example usingiodoacetimide, N-succinimidyl bromoacetate (SBA; SIB),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB),sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB),N-succinimidyl iodoacetate (SIA) or succinimidyl3-[bromoacetamido]proprionate (SBAP)). Preferably, the cyanate ester(optionally made by COAP chemistry) is coupled with hexane diamine oradipic acid dihydrazide (AOH) and the amino-derivatized saccharide isconjugated to the carrier protein using carbodiimide (e.g., EOAC or EOC)chemistry via a carboxyl group on the protein carrier. Such conjugatesare described for example in WO 93/15760, WO 95/08348 and WO 96/129094.

The conjugation of the activated capsular polysaccharides and thecarrier proteins may be achieved, for example, by reductive amination,as described, for example, in U.S. Patent Appl. Pub. Nos. 2006/0228380,2007/0231340, 2007/0184071 and 2007/0184072, WO 2006/110381, WO2008/079653, and WO 2008/143709, all of which are incorporated byreference in their entireties. For example, the activated capsularpolysaccharides and the carrier protein may be reacted with a reducingagent to form a conjugate. Reducing agents which are suitable includeborohydrides, such as sodium cyanoborohydride, borane-pyridine, sodiumtriacetoxyborohydride, sodium borohydride, or borohydride exchangeresin. At the end of the reduction reaction, there may be unreactedaldehyde groups remaining in the conjugates. The unreacted aldehydegroups may be capped using a suitable capping agent, such as sodiumborohydride (NaBH4). In an embodiment, the reduction reaction is carriedout in aqueous solvent. In another embodiment the reaction is carriedout in aprotic solvent. In an embodiment, the reduction reaction iscarried out in DMSO (dimethylsulfoxide) or in DMF (dimethylformamide)solvent.

The activated capsular polysaccharides may be conjugated directly to thecarrier protein or indirectly through the use of a spacer or linker,such as a bifunctional linker. The linker is optionallyheterobifunctional or homobifunctional, having for example a reactiveamino group and a reactive carboxylic acid group, 2 reactive aminogroups or two reactive carboxylic acid groups.

Other suitable techniques for conjugation use carbodiimides, hydrazides,active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide,S-NHS, EOC, TSTU, as described, for example, in International PatentApplication Publication No. WO 98/42721. Conjugation may involve acarbonyl linker which may be formed by reaction of a free hydroxyl groupof the saccharide with 1,1′-carbonyldiimidazole (CDI) (see Bethell etal. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al. (1981) J.Chromatogr. 218:509-518) followed by reaction with a protein to form acarbamate linkage. This may involve reduction of the anomeric terminusto a primary hydroxyl group, optional protection/deprotection of theprimary hydroxyl group, reaction of the primary hydroxyl group with CDIto form a CDI carbamate intermediate and coupling the CDI carbamateintermediate with an amino group on a protein.

The ratio of polysaccharide to carrier protein for pneumococcalconjugate vaccines is typically in the range 0.3-3.0 (w/w) but can varywith the serotype. The ratio can be determined either by independentmeasurement of the amounts of protein and polysaccharide present, or bymethods that give a direct measure of the ratio known in the art.Methods include ¹H NMR spectroscopy or the use of SEC-HPLC-UV/RI withdual monitoring (e.g. refractive index and UV, for total material andprotein content respectively) can profile the saccharide/protein ratioover the size distribution of conjugates as well as by SEC-HPLC-MALLS orMALDI-TOF-MS.

The polysaccharide-protein conjugates thus obtained may be purified andenriched by a variety of methods. These methods includeconcentration/diafiltration, column chromatography, and depthfiltration. The purified polysaccharide-protein conjugates are combinedto formulate a mixed carrier, 16-valent pneumococcal conjugatecomposition, which can be used as a vaccine.

Formulation of a vaccine composition can be accomplished usingart-recognized methods. A vaccine composition is formulated to becompatible with its intended route of administration. The individualpneumococcal capsular polysaccharide-protein conjugates can beformulated together with a physiologically acceptable vehicle to preparethe composition. Examples of such vehicles include, but are not limitedto, water, buffered saline, polyols (e.g., glycerol, propylene glycol,liquid polyethylene glycol) and dextrose solutions.

In some embodiments, the mixed carrier, 16-valent pneumococcal conjugatecomposition further comprises an adjuvant. Adjuvants can include asuspension of minerals (alum, aluminum salts, such as aluminumhydroxide, aluminum phosphate, aluminum sulfate, aluminumhydroxyphosphate sulfate, etc.) on which antigen is adsorbed; orwater-in-oil emulsion in which antigen solution is emulsified in mineraloil (for example, Freund's incomplete adjuvant), sometimes with theinclusion of killed mycobacteria (Freund's complete adjuvant) to furtherenhance antigenicity. Immunostimulatory oligonucleotides (such as thoseincluding a CpG motif) can also be used as adjuvants (for example, seeU.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116;6,339,068; 6,406,705; and 6,429,199). Adjuvants also include biologicalmolecules, such as lipids and costimulatory molecules. Exemplarybiological adjuvants include AS04 [9], IL-2, RANTES, GM-CSF, TNF-α,IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

In some embodiments, the adjuvant is an aluminum-based adjuvant.

In particular embodiments, the adjuvant is selected from the groupconsisting of aluminum phosphate, aluminum sulfate, and aluminumhydroxide.

In particular embodiments, the adjuvant is aluminum phosphate.

In some embodiments, the composition is for use as a vaccine against aninfection of Streptococcus pneumoniae.

Prophylactic Methods and Uses

In one aspect, this disclosure provides a vaccine comprising a mixedcarrier, 16-valent pneumococcal conjugate composition and apharmaceutically acceptable excipient. In some embodiment, thepharmaceutically acceptable excipient comprises at least a buffer, suchas a succinate buffer, a salt, such as sodium chloride, and/or a surfaceactive agent, such as a polyoxyethylene sorbitan ester (e.g.,polysorbate 80). In some embodiments, the capsular polysaccharides fromserotypes 1 and 5 are conjugated to the tetanus toxoid, and the capsularpolysaccharides from serotypes 3, 4, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A,19F, 22F, 23F, and 33F are conjugated to CRM₁₉₇. In another embodiment,the capsular polysaccharides from serotypes 1 and 3 are conjugated tothe tetanus toxoid, and the capsular polysaccharides from serotypes 4,5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and 33F areconjugated to CRM₁₉₇. In yet another embodiment, the capsularpolysaccharides from serotypes 3 and 5 are conjugated to the tetanustoxoid, and the capsular polysaccharides from serotypes 1, 4, 6A, 6B,7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and 33F are conjugated toCRM₁₉₇.

In some embodiments, the vaccine elicits a protective immune response ina human subject against disease caused by Streptococcus pneumoniaeinfection.

According to a further aspect, this disclosure provides a method forprophylaxis of Streptococcus pneumoniae infection or disease, the methodcomprising administering to a human subject a prophylactically effectiveamount of a mixed carrier, 16-valent pneumococcal conjugate compositionor a vaccine comprising the same. The mixed carrier, 16-valentpneumococcal conjugate composition or vaccine comprising the same may beadministered by any route, including, for example, by a systemic ormucosal route, as described below in further detail.

In certain embodiments, the human subject is an elderly subject and thedisease is pneumonia or invasive pneumococcal disease (IPD). In certainembodiments, the elderly subject is at least 50 years old. In otherembodiments, the elderly subject is at least 55 years old. In yet otherembodiments, the elderly subject is at least 60 years old.

In other embodiments, the human subject is an infant and the disease ispneumonia, invasive pneumococcal disease (IPD), or acute otitis media(AOM). In certain embodiments, the infant is 0-2 years. In otherembodiments, the infant is 2 to 15 months.

In yet another embodiment, the human subject is 6 weeks to 17 years ofage and the disease is pneumonia, invasive pneumococcal disease (IPD) oracute otitis media (AOM). In certain embodiments, the human subject is 6weeks to 5 years of age. In other embodiments, the human subject is 5 to17 years of age.

The amount of conjugate in each vaccine dose or the prophylacticallyeffective amount of the mixed carrier, multivalent pneumococcalconjugate composition may be selected as an amount that inducesprophylaxis without significant, adverse effects. Such an amount canvary depending upon the pneumococcal serotype. Generally, each dose mayinclude about 0.1 μg to about 100 μg of polysaccharide, specifically,about 0.1 to 10 μg, and, more specifically, about 1 μg to about 5 μg.Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. For example, the amount for vaccination ofa human subject can be determined by extrapolating an animal testresult. In addition, the dose can be determined empirically.

In some embodiments, the vaccine or the mixed carrier, 16-valentpneumococcal conjugate composition may be a single 0.5 ml doseformulated to contain about 1 μg to about 5 μg of each capsularpolysaccharide; about 1 μg to about 25 μg of TT; about 20 μg to about 75μg of CRM₁₉₇, and optionally about 0.1 mg to about 0.5 mg of elementalaluminum adjuvant. In some embodiments, the vaccine or the mixedcarrier, 16-valent pneumococcal conjugate composition may be a single0.5 ml dose formulated to contain about 2 μg to about 2.5 μg of eachcapsular polysaccharide except serotype 6B; about 4 μg to about 5 μg ofthe capsular polysaccharide of serotype 6B; about 5 μg to about 15 μg ofTT; about 30 μg to about 60 μg of CRM₁₉₇; and optionally about 0.1 mg toabout 0.2 mg of elemental aluminum adjuvant. In certain embodiments, themixed carrier, 16-valent pneumococcal conjugate composition or vaccinecomprising the same further comprises sodium chloride and sodiumsuccinate buffer as excipients.

In some embodiments, the mixed carrier, 16-valent pneumococcal conjugatecomposition may be formulated into a liquid formulation in which each ofthe pneumococcal capsular polysaccharides of serotypes 1 and 3 isconjugated to TT and each of the pneumococcal capsular polysaccharidesserotypes 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and33F is conjugated to CRM₁₉₇. Each 0.5 mL dose may be formulated into aliquid containing: about 2.2 μg of each polysaccharide, except for 6B atabout 4.4 μg; about 10 μg to about 15 μg of TT carrier protein (only forthe serotypes 1 and 3) and about 40 μg to about 50 μg of CRM₁₉₇ carrierprotein; 0.125 mg of elemental aluminum (0.5 mg aluminum phosphate) asan adjuvant; and sodium chloride and sodium succinate buffer asexcipients.

In some embodiments, the mixed carrier, 16-valent pneumococcal conjugatecomposition may be formulated into a liquid formulation in which each ofthe pneumococcal capsular polysaccharides of serotypes 1 and 5 isconjugated to TT and each of the pneumococcal capsular polysaccharidesserotypes 3, 4, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and33F is conjugated to CRM₁₉₇. Each 0.5 mL dose may be formulated into aliquid containing: about 2.2 μg of each saccharide, except for 6B atabout 4.4 μg; about 5 μg to about 10 μg of TT carrier protein (only forthe serotypes 1 and 5) and about 40 μg to about 50 μg of CRM₁₉₇ carrierprotein; about 0.125 mg of elemental aluminum (0.5 mg aluminumphosphate) adjuvant; and sodium chloride and sodium succinate buffer asexcipients.

In some embodiments, the mixed carrier, 16-valent pneumococcal conjugatecomposition may be formulated into a liquid formulation in which each ofthe pneumococcal capsular polysaccharides of serotypes 3 and 5 isconjugated to TT and each of the pneumococcal capsular polysaccharidesserotypes 1, 4, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and33F is conjugated to CRM₁₉₇. Each 0.5 mL dose may be formulated into aliquid containing: about 2.2 μg of each saccharide, except for 6B atabout 4.4 μg; about 10 μg to about 15 μg of TT carrier protein (only forthe serotypes 3 and 5) and about 40 μg to about 50 μg of CRM₁₉₇ carrierprotein; about 0.125 mg of elemental aluminum (0.5 mg aluminumphosphate) adjuvant; and sodium chloride and sodium succinate buffer asexcipients.

In some embodiments, the liquid formulation may be filled into a singledose syringe without a preservative. After shaking, the liquidformulation becomes a vaccine that is a homogeneous, white suspensionready for intramuscular administration.

The mixed carrier, 16-valent pneumococcal conjugate composition can beadministered in a single injection or as part of an immunization series.For example, the mixed carrier, 16-valent pneumococcal conjugatecomposition can be administered 2, 3, 4, or more times at appropriatelyspaced intervals, such as, a 1, 2, 3, 4, 5, or 6 month interval or acombination thereof. In some embodiments, the mixed carrier, 16-valentpneumococcal conjugate composition is administered to an infant 4 timeswithin the first 15 months of birth, including, for example, at about 2,3, 4, and 12-15 months of age; at about 3, 4, 5, and 12-15 months ofage; or at about 2, 4, 6, and 12 to 15 months of age. This first dosecan be administered as early as 6 weeks of age. In another embodiment,the mixed carrier, 16-valent pneumococcal conjugate composition isadministered to an infant 3 times within the first 15 months of birth,including, for example, at about 2, 4, and 11-12 months.

The mixed carrier, multivalent pneumococcal conjugate composition mayalso include one or more proteins from Streptococcus pneumoniae.Examples of Streptococcus pneumoniae proteins suitable for inclusioninclude those identified in International Patent ApplicationWO02/083855, as well as those described in International PatentApplication WO02/053761.

The mixed carrier, 16-valent pneumococcal conjugate composition can beadministered to a subject via one or more administration routes known toone of ordinary skill in the art such as a parenteral, transdermal, ortransmucosal, intranasal, intramuscular, intraperitoneal,intracutaneous, intravenous, or subcutaneous route and be formulatedaccordingly. The mixed carrier, 16-valent pneumococcal conjugatecomposition can be formulated to be compatible with its intended routeof administration.

In some embodiments, the mixed carrier, 16-valent pneumococcal conjugatecomposition can be administered as a liquid formulation byintramuscular, intraperitoneal, subcutaneous, intravenous,intraarterial, or transdermal injection or respiratory mucosalinjection. The mixed carrier, 16-valent pneumococcal conjugatecompositions can be formulated in liquid form or in a lyophilized form.In some embodiments, injectable compositions are prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. In some embodiments, injection solutions andsuspensions are prepared from sterile powders or granules. Generalconsiderations in the formulation and manufacture of pharmaceuticalagents for administration by these routes may be found, for example, inRemington's Pharmaceutical Sciences, 19^(th) ed., Mack Publishing Co.,Easton, Pa., 1995; incorporated herein by reference. At present the oralor nasal spray or aerosol route (e.g., by inhalation) are most commonlyused to deliver therapeutic agents directly to the lungs and respiratorysystem. In some embodiments, a mixed carrier, 16-valent pneumococcalconjugate composition is administered using a device that delivers ametered dosage of composition. Suitable devices for use in deliveringintradermal pharmaceutical compositions described herein include shortneedle devices such as those described in U.S. Pat. Nos. 4,886,499,5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496,5,417,662 (all of which are incorporated herein by reference).Intradermal compositions may also be administered by devices which limitthe effective penetration length of a needle into the skin, such asthose described in WO1999/34850, incorporated herein by reference, andfunctional equivalents thereof. Also suitable are jet injection deviceswhich deliver liquid vaccines to the dermis via a liquid jet injector orvia a needle which pierces the stratum corneum and produces a jet whichreaches the dermis. Jet injection devices are described for example inU.S. Pat. Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912,5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163,5,312,335, 5,503,627, 5,064,413, 5,520,639, 4,596,556, 4,790,824,4,941,880, 4,940,460, WO1997/37705, and WO1997/13537 (all of which areincorporated herein by reference). Also suitable are ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis. Additionally, conventional syringes may be used in the classicalmantoux method of intradermal administration.

Preparations for parenteral administration include sterile aqueous ornonaqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol, oilssuch as olive oil, and injectable organic esters such as ethyl oleate.Examples of oil include vegetable or animal oil, peanut oil, soybeanoil, olive oil, sunflower oil, liver oil, synthetic oil such as marineoil, and lipids obtained from milk or eggs. Aqueous carriers includewater, alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like.

The mixed carrier, 16-valent pneumococcal conjugate composition can beformulated in the form of a unit dose vial, multiple dose vial, orpre-filled syringe. A pharmaceutically acceptable carrier for a liquidformulation includes aqueous or nonaqueous solvent, suspension,emulsion, or oil. The composition may be isotonic, hypertonic, orhypotonic. However, it is desirable that the composition for infusion orinjection is basically isotonic. Thus, isotonicity or hypertonicity maybe advantageous for storage of the composition. When the composition ishypertonic, the composition can be diluted to isotonicity beforeadministration. A tonicity agent may be ionic tonicity agent such assalt or non-ionic tonicity agent such as carbohydrate. The ionictonicity agent includes, but is not limited to, sodium chloride, calciumchloride, potassium chloride, and magnesium chloride. The nonionictonicity agent includes, but is not limited to, sorbitol and glycerol.Preferably, at least one pharmaceutically acceptable buffer is included.For example, when the composition is an infusion or injection, it ispreferable to be formulated in a buffer with a buffering capacity at pH4 to pH 10, such as pH 5 to pH 9, or, pH 6 to pH 8. The buffer may beselected from those suitable for United States Pharmacopeia (USP). Forexample, the buffer can be selected from the group consisting of amonobasic acid, such as acetic acid, benzoic acid, gluconic acid,glyceric acid, and lactic acid; a dibasic acid, such as aconitic acid,adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid,succinic acid, and tartaric acid; a polybasic acid such as citric acidand phosphoric acid; and a base such as ammonia, diethanolamine,glycine, triethanolamine, and TRIS.

The mixed carrier, 16-valent pneumococcal conjugate composition maycomprise a surface active agent. Examples of the surface active agentinclude, but are not limited to, polyoxyethylene sorbitan ester(generally referred to as Tweens), in particular, polysorbate 20 andpolysorbate 80; copolymers (such as DOWFAX) of ethylene oxide (EO),propylene oxide (PO), butylenes oxide (BO); octoxynols with differentrepeats of ethoxy(oxy-1,2-ethanediyl) group, in particular, octoxynol-9(Triton-100); ethylphenoxypolyethoxyethanol (IGEPAL CA-630/NP-40);phospholipid such as lecithin; nonylphenol ethoxylate such as TERGITOLNP series; lauryl, cetyl, stearyl, oleyl alcohol-derived polyoxyethylenefatty ether (Brij surfactant), in particular, triethyleneglycolmonolauryl ether (Brij 30); sorbitan ether known as SPAN, in particular,sorbitan trioleate (Span 85) and sorbitan monolaurate.

Mixtures of surface active agents such as Tween 80/Span 85 can be used.A combination of polyoxyethylene sorbitan ester such as Tween 80 andoctoxynol such as Triton X-100 is also suitable. A combination ofLaureth 9 and Tween and/or octoxynol is also advantageous. Preferably,the amount of polyoxyethylene sorbitan ester (such as Tween 80) includedmay be 0.01% to 1% (w/v), 0.01% to 0.1% (w/v), 0.01% to 0.05% (w/v), orabout 0.02%; the amount of octylphenoxy polyoxyethanol or nonylphenoxypolyoxyethanol (such as Triton X-100) included may be 0.001% to 0.1%(w/v), in particular 0.005% to 0.02%; and the amount of polyoxyethyleneether (such as Laureth 9) included may be 0.1% to 20% (w/v), possibly0.1% to 10%, in particular 0.1% to 1% or about 0.5%.

In some embodiments, the mixed carrier, 16-valent pneumococcal conjugatecomposition may be delivered via a release control system. For example,intravenous infusion, transdermal patch, liposome, or other routes canbe used for administration. In one aspect, macromolecules such asmicrosphere or implant can be used.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theinvention.

Examples Example 1. Preparation of S. pneumoniae CapsularPolysaccharides

Cultivation of S. pneumoniae and purification of capsularpolysaccharides were conducted as known to one of skill in the art. S.pneumoniae serotypes were obtained from the American Type CultureCollection (ATCC) (serotype 1: ATCC No. 6301; serotype 3: ATCC No. 6303;serotype 4: ATCC No. 6304; serotype 5: ATCC No. 6305; serotype 6A: ATCCNo. 6306; serotype 6B: ATCC No. 6326; serotype 7F: ATCC No. 10351;serotype 9V: ATCC No. 10368; serotype 12F: ATCC No. 6312; serotype 14:ATCC No. 6314; serotype 18C: ATCC No. 10356; serotype 19A: ATCC No.10357; serotype 19F: ATCC No. 6319; serotype 22F: ATCC No. 6322;serotype 23F: ATCC No. 6323; serotype 33F: ATCC No. 10370). S.pneumoniae were characterized by capsules and immobility, Gram-positive,lancet-shaped diplococcus, and alpha hemolysis in a blood agar medium.Serotypes were identified by Quelling test using specific anti-sera(U.S. Pat. No. 5,847,112).

Preparation of Cell Banks

Several generations of seed stocks were generated in order to expand thestrains and remove components of animal origin (generations F1, F2, andF3). Two additional generations of seed stocks were produced. The firstadditional generation was cultured from an F3 vial, and the subsequentgeneration was cultured from a vial of the first additional generation.Seed vials were stored frozen (below −70° C.) with synthetic glycerol asa cryopreservative. For cell bank preparation, all cultures were grownin a soy-based medium. Prior to freezing, cells were concentrated bycentrifugation, spent medium was removed, and cell pellets werere-suspended in a fresh medium containing a cryopreservative (such assynthetic glycerol).

Culturing and Harvesting

Cultures from the working cell bank were inoculated into seed bottlescontaining a soy-based medium and cultured. After the target opticaldensity (absorbance) was reached, the seed bottle was used to inoculatea fermentor containing the soy-based medium. The culturing wasterminated when an optical density value started to be maintainedconstant. After terminating the culturing, sodium deoxycholate was addedto the culture to lyse the cells. The resulting fermentor contents werecooled, and protein precipitation was induced. Then, the mixture wascentrifuged to remove precipitated proteins and cell debris.

Purification

The solution obtained from the centrifugation was filtered through adepth filter to remove the proteins and cell debris that had notprecipitated in the centrifugation. The filtrate was concentrated on a100 kDa MW membrane and the concentrate was diafiltered with 10 volumesof a 25 mM sodium phosphate buffer (pH 7.2) to obtain a sample. Thesample was filtered to collect a supernatant from which polysaccharideswere precipitated and filtered. The filtrate was concentrated on a 30kDa membrane, and the concentrate was diafiltered using about 10 volumesof triple distilled water. After performing the diafiltration, theremaining solution was filtered through a 0.2 μm filter. An in-processcontrol test was performed on the filtrate (appearance, remainingproteins, remaining nucleic acids, endotoxins, molecular weights, andthe total amount of polysaccharides). The concentrate was sterilefiltered and stored at −20° C.

Example 2. Preparation of Conjugate of S. pneumoniae CapsularPolysaccharide and Carrier Protein

Polysaccharides of different serotypes were activated followingdifferent pathways and then conjugated to a carrier protein, CRM₁₉₇ orTT. Specifically, conjugates were prepared by conjugating each of thecapsular polysaccharides of all serotypes to CRM₁₉₇ and by conjugatingeach of the capsular polysaccharides of the serotypes 1, 3, and 5 to TT.The activation process includes reduction of the size of each capsularpolysaccharide to the target molecular weight, chemical activation, andbuffer exchange via ultrafiltration. The conjugates were purified usingultrafiltration and finally filtered through 0.2 μm filter. The processparameters such as pH, temperature, concentration, and time were asfollows.

(1) Activation Process

Step 1: Hydrolysis

Reductive amination is a known method for conjugating polymers in whichan amide bond is formed between a primary amine (—NH₂) group of aprotein and an aldehyde of a saccharide. However, since S. pneumoniaepolysaccharides do not have any aldehyde groups, an aldehyde group isadded to the pneumococcal capsular polysaccharide. A diol structure of amonosaccharide can be oxidized by sodium periodate (NaIO₄) to form analdehyde group. The capsular polysaccharides from serotypes 1, 3, 4, 6A,12F, 14, 18C, 22F, and 33F were pre-treated as follows.

In the case of the serotype 1, sodium hydroxide (at a final baseconcentration of 0.05 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 50±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and hydrochloric acid was added thereto to a final pH of 6.0±0.1,thereby stopping hydrolysis.

In the case of the serotype 3, hydrochloric acid (at a final acidconcentration of 0.01 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 60±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and 0.1M sodium phosphate was added thereto to a final pH of6.0±0.1, thereby stopping hydrolysis.

In the case of the serotype 4, hydrochloric acid (at a final acidconcentration of 0.1 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 45±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and 1M sodium phosphate was added thereto to a final pH of 6.0±0.1,thereby stopping hydrolysis.

In the case of the serotype 6A, glacial acetic acid (at a final acidconcentration of 0.1 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 60±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and 1M sodium hydroxide was added thereto to a final pH of 6.0±0.1.

In the case of the serotype 12F, hydrochloric acid (at a final acidconcentration of 0.01 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 70±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and 0.1M sodium phosphate was added thereto to a final pH of thesolution of 6.0±0.1, thereby stopping hydrolysis.

In the case of the serotypes 14 and 18C, glacial acetic acid (at a finalacid concentration of 0.2 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at about 91-96° C. Thesolution was then cooled to a temperature in a range of about 21° C. toabout 25° C., and 1M sodium phosphate was added thereto so that a finalpH of the solution was 6.0±0.1.

In the case of the serotypes 22F and 33F, hydrochloric acid (at a finalacid concentration of 0.01 M) was added to a solution of the capsularpolysaccharide, and the solution was incubated at 60±2° C. The solutionwas then cooled to a temperature in a range of about 21° C. to about 25°C., and 0.1M sodium phosphate was added thereto to a final pH of6.0±0.1, thereby stopping hydrolysis.

Each of the obtained capsular polysaccharides was diluted in water forinjection (WFI), sodium acetate, and sodium phosphate to a finalconcentration between about 1.0 mg/mL and about 2.0 mg/mL.

Step 2: Periodate Reaction

The sodium periodate molar equivalent for each pneumococcal saccharideactivation was determined using total saccharide content. With thoroughmixing, the oxidation reaction was allowed to proceed for 16 to 20 hoursat 21° C. to 25° C. for all serotypes except for 1, 7F, and 19F, forwhich the temperature was 10° C. or less. During the conjugationprocess, maintaining an aldehyde concentration at an appropriate levelprovides for consistent and stable production of conjugates. A degree ofproduction of the aldehyde is determined by a ratio between aconcentration of the produced aldehyde and a concentration ofsaccharide, and this degree is related to a degree of oxidation (Do)which is set for each serotype as shown in Table 2 and Table 3.

TABLE 2 Range of Do for all serotypes to be conjugated to CRM₁₉₇Serotype Range of Do Serotype 1 4 to 10 Serotype 3 2 to 8 Serotype 4 1to 5 Serotype 5 2 to 6 Serotype 6A 5 to 15 Serotype 6B 7 to 13 Serotype7F 2 to 8 Serotype 9V 4 to 9 Serotype 12F 1 to 9 Serotype 14 6 to 13Serotype 18C 6 to 14 Serotype 19A 7 to 13 Serotype 19F 6 to 12 Serotype22F 1 to 16 Serotype 23F 6 to 14 Serotype 33F 1 to 15

TABLE 3 Range of Do for serotypes 1, 3, and 5 to be conjugated to TTSerotype Range of Do Serotype 1 1 to 15 Serotype 3 2 to 14 Serotype 5 1to 15

Step 3: Ultrafiltration

The oxidized saccharide was concentrated and diafiltered with WFI on a100 kDa MWCO ultrafilter (30 kDa ultrafilter for serotype 1 and 5 kDaultrafilter for serotype 18C). Diafiltration was conducted using 0.9%sodium chloride solution for serotype 1, 0.01 M sodium acetate buffer(pH 4.5) for serotypes 7F and 23F and 0.01 M sodium phosphate buffer (pH6.0) for serotype 19F. The permeate was discarded, and the retentate wasfiltered through a 0.2 μm filter.

Step 4: Lyophilization

For capsular polysaccharides of serotypes 3, 4, 5, 9V, 14, 22F, and 33Fthat are to be conjugated to a carrier protein by using an aqueoussolvent, a mixed solution of polysaccharides and carrier protein wasprepared without addition of sucrose, lyophilized, and then stored at−25° C.±5° C.

For capsular polysaccharides of serotypes 1 and 18C that are to beconjugated to a carrier protein by using an aqueous solvent,polysaccharides and carrier protein were independently prepared withoutaddition of sucrose, lyophilized, and then stored at −25° C.±5° C.

For capsular polysaccharides of serotypes 6A, 6B, 7F, 19A, 19F, and 23Fthat are to be conjugated to a carrier protein by using a DMSO solvent,a predetermined amount of sucrose to reach a final sucrose concentrationof 5%±3% was added to the activated saccharides, and the samples wereindependently prepared, lyophilized, and then stored at −25° C.±5° C.

For capsular polysaccharide of serotype 12F, a predetermined amount ofsucrose to reach a final sucrose concentration of 10%±5% was added tothe activated saccharide, and the sample were filled into glass bottles,lyophilized, and then stored at −25° C.±5° C.

(2) Conjugation Process

Aqueous conjugation was conducted for serotypes 1, 3, 4, 5, 9V, 14, 18C,22F, and 33F, and DMSO conjugation was conducted for serotypes 6A, 6B,7F, 12F, 19A, 19F, and 23F. Each of the capsular polysaccharides wasconjugated to a carrier protein at a ratio of 0.2 to 2:1.

Step 1: Dissolution

Aqueous Conjugation

For serotypes 1, 3, 4, 5, 9V, 14, 18C, 22F, and 33F, the lyophilizedsample was thawed and equilibrated at room temperature. The lyophilizedsample was reconstituted to a reaction concentration by using a sodiumphosphate buffer solution at 23±2° C. at a ratio set for each serotype.

Dimethyl Sulfoxide (DMSO) Conjugation

For serotypes 6A, 6B, 7F, 12F, 19A, 19F, and 23F, the lyophilized samplewas thawed, equilibrated at room temperature, and reconstituted in DMSO.

Step 2: Conjugation Reaction

Aqueous Conjugation

For serotypes 1, 3, 4, 5, 9V, 14, 18C, 22F, and 33F to be conjugated toCRM₁₉₇, the conjugation reaction was initiated by adding the sodiumcyanoborohydride solution (100 mg/mL) to 1.0 to 1.4 moles sodiumcyanoborohydride per mole of saccharide. However, for serotypes 1 and 3,the reaction was initiated by adding the sodium cyanoborohydridesolution to 0.5 moles sodium cyanoborohydride per mole of saccharide.

For serotypes 1, 3, and 5 to be conjugated to TT, the conjugationreaction was initiated by adding the sodium cyanoborohydride solution(100 mg/mL) to 1.0 to 1.4 moles sodium cyanoborohydride per mole ofsaccharide, except for serotype 1 to 0.5 moles sodium cyanoborohydrideper mole of saccharide.

The reaction mixture was incubated at 23° C. to 37° C. for 44 to 106hours. The reaction temperature and time were adjusted by serotype. Thetemperature was then reduced to 23±2° C. and sodium chloride 0.9% wasadded to the reactor. Sodium borohydride solution (100 mg/mL) was addedto achieve 1.8 to 2.2 molar equivalents of sodium borohydride per moleof saccharide. The mixture was incubated at 23±2° C. for 3 to 6 hours.This procedure reduced any unreacted aldehydes present on thesaccharides. Then, the mixture was diluted with sodium chloride 0.9% andthe diluted conjugation mixture was filtered using a 0.8 or 0.45 μmpre-filter.

DMSO Conjugation

For capsular polysaccharides of serotypes 6A, 6B, 7F, 12F, 19A, 19F, and23F, the conjugation reaction was initiated by adding the sodiumcyanoborohydride solution (100 mg/mL) to a ratio of 0.8 to 1.2 molarequivalents of sodium cyanoborohydride per one mole of activatedsaccharide. WFI was added to the reaction mixture to a targetconcentration of 1% (v/v), and the mixture was incubated for 12 to 26hours at 23±2° C. 100 mg/mL of a sodium borohydride solution (typical1.8 to 2.2 molar equivalents sodium borohydride per mole activatedsaccharide) and WFI (target 5% v/v) were added to the reaction and themixture was incubated for 3 to 6 hours at 23±2° C. This procedurereduced any unreacted aldehydes present on the saccharides. Then, thereaction mixture was diluted with sodium chloride 0.9%, and the dilutedconjugation mixture was filtered using a 0.8 or 0.45 μm pre-filter.

Step 3: Ultrafiltration

The diluted conjugate mixture was concentrated and diafiltered on a 100kDa MWCO ultrafiltration filter or a 300 kDa MWCO ultrafiltration filterwith a minimum of 15 volumes of 0.9% sodium chloride or buffer. Also, atype and pH of the buffer used in the process varied depending on eachof the serotypes.

Step 4: Sterile Filtration

The retentate after the ultrafiltration was sterile filtered (0.2 μm),and in-process controls (appearance, free protein, free saccharide,molecular size distribution, sterility, saccharide content, proteincontent, pH, endotoxin, residual cyanide, residual DMSO, saccharideidentity, TT identity, and CRM₁₉₇ identity) were performed on thefiltered conjugates. The final concentrate was refrigerated and storedat 2° C. to 8° C.

Example 3. Formulation of Multivalent Pneumococcal Conjugate Vaccine

The desired volumes of final bulk concentrates obtained from Example 2were calculated based on the batch volume and the bulk saccharideconcentrations. After the 0.85% sodium chloride (physiological saline),polysorbate 80, and succinate buffer were added to the pre-labeledformulation vessel, bulk concentrates were added. The preparation wasthen thoroughly mixed and sterile filtered through a 0.2 μm membrane.The formulated bulk was mixed gently during and following the additionof bulk aluminum phosphate. The pH was checked and adjusted ifnecessary. The formulated bulk product was stored at 2° C. to 8° C. Thefollowing four types of multivalent pneumococcal conjugate vaccineformulations were prepared and named PCV16-CRM₁₉₇, PCV16-13TT,PCV16-15TT, and PCV16-35TT, respectively.

PCV16-CRM₁₉₇ included polysaccharide-conjugates prepared by conjugatingeach of polysaccharides of 16 serotypes (serotypes 1, 3, 4, 5, 6A, 6B,7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and 33F) to CRM₁₉₇;

PCV16-13TT included polysaccharide-conjugates prepared by conjugatingeach polysaccharide of the serotypes 1 and 3 to TT and eachpolysaccharide of the serotypes 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A,19F, 22F, 23F, and 33F to CRM₁₉₇;

PCV16-15TT included polysaccharide-conjugates prepared by conjugatingeach polysaccharide of the serotypes 1 and 5 to TT and eachpolysaccharide of the serotypes 3, 4, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A,19F, 22F, 23F, and 33F to CRM₁₉₇; and

PCV16-35TT included polysaccharide-conjugates prepared by conjugatingeach polysaccharide of the serotypes 3 and 5 to TT and eachpolysaccharide of the serotypes 1, 4, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A,19F, 22F, 23F, and 33F to CRM₁₉₇.

The PCV16-13TT and PCV16-35TT vaccine composition obtained included 2.2μg of each saccharide, except for serotype 6B at 4.4 μg; 10 ug to 15 ugof TT (for serotypes 1 and 3) and 40 μg to 50 μg of CRM₁₉₇; 0.125 mg ofelemental aluminum (0.5 mg aluminum phosphate) adjuvant; 4.25 mg ofsodium chloride; about 295 μg of a succinate buffer solution; and about100 μg of polysorbate 80 in the total of 0.5 ml dose. The PCV16-15TTcomposition in a total dose of 0.5 mL included 5 μg to 10 μg of TT and40 μg to 50 μg CRM₁₉₇, and, respectively, with the other components andcontents thereof identical to those of PCV16-13TT and PCV16-35TT.

Example 4. Immunogenicity of Multivalent Pneumococcal Conjugate Vaccine

The mixed carrier, multivalent pneumococcal vaccines, PCV16-CRM₁₉₇,PCV16-13TT, PCV16-15TT, and PCV16-35TT prepared in Example 3, weretested for the ability to induce an immunogenic response in rabbits.These immunogenic effects were characterized by antigen-specific ELISAfor serum IgG concentrations and by opsonophagocytic assay (OPA) forantibody function. New Zealand White rabbits were immunizedintramuscularly at week 0 and week 3 with a dose of 5% higher than theplanned human clinical dose of each polysaccharide (2.31 μg of eachpolysaccharide, except for 6B at 4.62 μg) in the formulation or thehuman dose (2.2 ug of each polysaccharide, except for 6B at 4.4 ug).Sera were sampled every 3 weeks post immunization. Both concentrationsshowed the same results.

A serotype-specific immune reaction with respect to PCV16-CRM₁₉₇,PCV16-13TT, PCV16-15TT, and PCV16-35TT compositions was evaluated by IgGELISA and a complement-mediated MOPA that measures a functionalantibody.

4-1. PCV16-CRM₁₉₇

Serotype specific IgG concentration measurement

Capsular polysaccharides (PnPs) for each serotype were coated on a96-well plate at 0.5 μg/well to 1 μg/well. An equivalent amount of serumwas sampled from each subject and was pooled by group. The serum poolwas serially diluted by 2.5 times with an antibody dilution buffercomprising Tween 20 and CWPS 5 μg/mL and then reacted at roomtemperature for 30 minutes. The plate was washed 5 times with a washingbuffer and then pre-adsorbed and diluted serum 50 μl was added to thecoated well plate, followed by incubation at room temperature for 2hours to 18 hours. The well plate was washed in the same way and thengoat anti-Rabbit IgG-alkaline phosphatase conjugates were added to eachwell, followed by incubation at room temperature for 2 hours. Plateswere washed as described above and 1 mg/mL p-nitrophenylamine buffer assubstrate was added to each well and then reacted at room temperaturefor 2 hours. The reaction was quenched by adding 50 μl of 3 M NaOH andabsorbances at 405 nm and 690 nm were measured. As a comparativeexample, the commercially available, 13-valent vaccine (PREVNAR13) wassubjected to the same procedure. The results are shown in Table 4.

TABLE 4 IgG concentration (U/mL) for 16 serotypes at 3 weeks aftersecondary immunization PCV16- Serotype PREVNAR13 CRM197  1 320.99 379.99 3 436.85 653.84  4 1820.49 1948.29  5 466.09 380.18  6A 1064.69 1643.6 6B 326.94 552.58  7F 1010.79 833.11  9V 715.40 433.33 12F — 120.44 14482.05 502.6 18C 1731.07 2915.55 19A 993.68 672.2 19F 863.32 1054.3 22F1.33 678.45 23F 329.11 185.97 33F 4.58 499.3

PCV16-CRM₁₉₇ was found to lead to good levels of serotype specific IgGconcentrations for all 16 serotypes. For PCV16-CRM₁₉₇, the serotypescommon to the PCV16-CRM₁₉₇ and PREVNAR13 showed serotype-specific IgGconcentrations equivalent to or higher than that of PREVNAR13 and eachof the newly added serotypes 12F, 22F, and 33F also showed a good levelof serotype specific IgG concentration.

Functional Immunogenicity Test (MOPA)

Antibody functions were evaluated by testing serum in a MOPA assay. S.pneumoniae MOPA strain stored at −70° C. or lower was diluted to thecorresponding final dilution fold so that a concentration of each strainwas about 50,000 CFU/mL. An equivalent amount of serum was sampled fromeach subject, pooled by group and 2-fold serially diluted so that 20 μlof serum remained in a U-bottom plate. After diluting the sample, 10 μlof the strain prepared for each serotype was mixed with the dilutedsample, and the mixture was allowed to react at room temperature for 30minutes so that S. pneumoniae and the antibody were well mixed. Amixture of pre-differentiated HL-60 cells and complement was added andreacted in a CO₂ incubator (37° C.) for 45 minutes. The temperature wasreduced to stop phagocytosis and 10 μl of the reaction solution wasspotted onto an agar plate pre-dried for 30 to 60 minutes, and thenallowed to be absorbed onto the plate for 20 minutes until drying. A 25mg/mL TTC stock solution was added to a prepared overlay agar, and anantibody appropriate for the corresponding strain was added thereto. Themixture was thoroughly mixed, and then about 25 mL of the mixture wasadded onto the plate and hardened for about 30 minutes. The completelyhardened plate was incubated in a CO₂ incubator (37° C.) for 12 to 18hours and then colonies were counted. MOPA titer was expressed as adilution rate at which 50% killings were observed. As a comparativeexample, the commercially-available, 13-valent vaccine (PREVNAR13) wassubjected to the same procedure. The results are shown in Table 5.

TABLE 5 MOPA titers for 16 serotypes at 3 weeks after secondaryimmunization PCV16- Serotype PREVNAR13 CRM197  1 128 128  3 512 1024  42048 2048  5 512 256  6A 4096 4096  6B 4096 4096  7F 2048 1024  9V 1024512 12F 4 1024 14 2048 1024 18C 1024 2048 19A 4096 2048 19F 2048 204822F 16 4096 23F 2048 1024 33F 32 1024

All serotypes showed an excellent level of functional immunogenicity inPCV16-CRM₁₉₇. For PCV16-CRM₁₉₇, the serotypes common to the PCV16-CRM₁₉₇and PREVNAR13 showed functional immunogenicity equivalent to or betterthan that of PREVNAR13 and each of the newly added serotypes 12F, 22F,and 33F also showed a high level of functional immunogenicity.

4-2. PCV16-13TT

The serotype specific IgG concentration and functional immunogenicitytiter were measured in the same manner as in 4-1, except that PCV16-13TTwas used instead of PCV16-CRM₁₉₇, and the results are shown as follows.

Serotype Specific IgG Concentration Measurement

TABLE 6 IgG concentration (U/mL) for 16 serotypes at 3 weeks aftersecondary immunization PCV16- Serotype PREVNAR13 13TT  1 276.92 844.48 3 539.40 2980.73  4 1000.76 1698.00  5 303.20 184.49  6A 533.35 532.02 6B 172.75 451.18  7F 726.27 3449.73  9V 647.71 725.14 12F 0.37 354.0014 254.59 582.61 18C 3266.87 5553.58 19A 681.62 1702.05 19F 528.771998.83 22F 0.74 1583.58 23F 576.63 367.71 33F 0.25 977.02

When the capsular polysaccharides of serotypes 1 and 3 were conjugatedto TT, they showed significantly increased levels of serotype specificIgG compared to that obtained when the serotypes were conjugated toCRM₁₉₇. Also, each of the capsular polysaccharides of serotypes 12F,22F, and 33F conjugated to CRM₁₉₇ showed a decent level of serotypespecific IgG concentration and serotypes 4, 6B, 7F, 14, 18C, 19A, and19F showed higher levels of serotype specific IgG concentrations than inPREVNAR13.

Functional Immunogenicity Test (MOPA)

TABLE 7 MOPA titers for 16 serotypes at 3 weeks after secondaryimmunization PCV16- Serotype PREVNAR13 13TT  1 102 645  3 813 3251  42580 3251  5 813 406  6A 4096 4096  6B 4096 6502  7F 2580 6502  9V 20483251 12F 4 1625 14 2048 4096 18C 4096 4096 19A 2580 5161 19F 2048 516122F 8 5161 23F 4096 3251 33F 4 3251

When the capsular polysaccharides of serotypes 1 and 3 were conjugatedto TT, the functional immunogenicity improved compared to that obtainedwhen they were conjugated to CRM₁₉₇. Also, each of the capsularpolysaccharides of serotypes 12F, 22F, and 33F conjugated to CRM₁₉₇showed excellent functional immunogenicity and each of the capsularpolysaccharides of serotypes 4, 6B, 7F, 9V, 14, 19A, and 19F showedbetter functional immunogenicity than in PREVNAR13.

4-3. PCV16-15TT

The serotype specific IgG concentration and functional immunogenicitytiter were measured in the same manner as in 4-1, except that PCV16-15TTwas used instead of PCV16-CRM₁₉₇, and the results are shown as follows.

Serotype Specific IgG Concentration Measurement

TABLE 8 IgG concentration (U/mL) for 16 serotypes at 3 weeks aftersecondary immunization PCV16- Serotype PREVNAR13 15TT  1 276.92 1083.23 3 539.40 901.37  4 1000.76 2655.28  5 303.20 2645.56  6A 533.35 1460.65 6B 172.75 603.87  7F 726.27 2285.92  9V 647.71 663.37 12F 0.37 303.9914 254.59 493.06 18C 3266.87 4075.62 19A 681.62 937.41 19F 528.771355.08 22F 0.74 1874.55 23F 576.63 607.40 33F 0.25 880.54

When the capsular polysaccharides of serotypes 1 and 5 were conjugatedto TT, the serotype specific IgG concentration significantly increasedcompared to that obtained when they were conjugated to CRM₁₉₇. Also,each of the capsular polysaccharides of serotypes 12F, 22F, and 33Fconjugated to CRM₁₉₇ showed a high level of serotype specific IgGconcentration and each of the capsular polysaccharides of serotypes 3,4, 6A, 6B, 7F, 14, 18C, 19A, and 19F showed a higher level of serotypespecific IgG concentration than in PREVNAR13.

Functional Immunogenicity Test (MOPA)

TABLE 9 MOPA titers for 16 serotypes at 3 weeks after secondaryimmunization PCV16- Serotype PREVNAR13 15TT  1 102 645  3 813 1290  42580 4096  5 813 4096  6A 4096 6502  6B 4096 6502  7F 2580 4096  9V 20481290 12F 4 1290 14 2048 2580 18C 4096 3251 19A 2580 2580 19F 2048 409622F 8 6502 23F 4096 3251 33F 4 2580

When the capsular polysaccharides of serotypes 1 and 5 were conjugatedto TT, the functional immunogenicity improved compared to that obtainedwhen they were conjugated to CRM₁₉₇. Also, each of the capsularpolysaccharides of serotypes 12F, 22F, and 33F conjugated to CRM₁₉₇showed excellent functional immunogenicity and each of the capsularpolysaccharides of serotypes 3, 4, 6A, 6B, 7F, and 19F showed a higherlevel of functional immunogenicity than in PREVNAR13.

4-4. PCV16-35TT

The serotype specific IgG concentration and functional immunogenicitytiter were measured in the same manner as in 4-1, except that PCV16-35TTwas used instead of PCV16-CRM₁₉₇, and the results are shown as follows.

Serotype Specific IgG Concentration Measurement

TABLE 10 IgG concentration (U/mL) for 16 serotypes at 3 weeks aftersecondary immunization PCV16- Serotype PREVNAR13 35TT  1 242.33 367.13 3 656.91 1837.36  4 1305.61 1786.84  5 408.78 1316.12  6A 737.55 957.85 6B 167.41 322.61  7F 808.75 1357.46  9V 775.28 966.22 12F 0.25 334.4514 320.12 1055.422 18C 2920.75 3665.59 19A 652.67 409.17 19F 411.07534.19 22F 1.15 1176.6 23F 742.55 408.88 33F 0.25 855.55

When the capsular polysaccharides of serotypes 3 and 5 were conjugatedto TT, the serotype specific IgG concentration significantly increasedcompared to that obtained when they were conjugated to CRM₁₉₇. Also,each of the capsular polysaccharides of the serotypes 12F, 22F, and 33Fconjugated to CRM₁₉₇ had good serotype IgG concentration and each of thecapsular polysaccharides of serotypes 1, 4, 6A, 6B, 7F, 9V, 14, 18C, and19F showed a higher level of serum specific IgG concentration than inPREVNAR13.

Functional Immunogenicity Test (MOPA)

TABLE 11 MOPA titers for 16 serotypes at 3 weeks after secondaryimmunization PCV16- Serotype PREVNAR13 35TT  1 128 256  3 512 2048  42048 4096  5 512 2048  6A 4096 4096  6B 4096 4096  7F 2048 2048  9V 10242048 12F 4 512 14 2048 4096 18C 1024 4096 19A 4096 4096 19F 2048 409622F 16 4096 23F 2048 2048 33F 32 2048

When the serotypes 3 and 5 were conjugated to TT, the functionalimmunogenicity improved compared to that obtained when they wereconjugated to CRM₁₉₇. Also, each of the capsular polysaccharides ofserotypes 12F, 22F, and 33F conjugated to CRM₁₉₇ showed excellentfunctional immunogenicity and each of the capsular polysaccharides ofserotypes 1, 4, 9V, 12F, 14, 18C, and 19F showed a higher level offunctional immunogenicity than in PREVNAR13.

These results show that mixed carrier, 16-valent pneumococcal capsularpolysaccharide conjugate compositions induce immunogenicity equivalentto or better than the single carrier, pneumococcal capsularpolysaccharide conjugate vaccine, PREVNAR13. They also unexpectedly showthat the antibody response to serotypes 1, 3, and/or 5 conjugated totetanus toxoid in the mixed carrier compositions were significantlyenhanced as compared to the antibody responses against the sameserotypes conjugated to CRM₁₉₇ in the single carrier PREVNAR 13 vaccine.In addition, they show that the mixed carrier, 16-valent pneumococcalcapsular polysaccharide conjugate compositions successfully induceantibody responses against the added serotypes, 12F, 22F, and 33F,providing broader serotype protection than the pneumococcal capsularpolysaccharide conjugate vaccines currently on the market.

While one or more exemplary embodiments have been described in thespecification, it will be understood by those of ordinary skill in theart that various changes in form and details may be made therein withoutdeparting from the spirit and scope of the inventive concept as definedby the following claims.

REFERENCES

The following references are cited in the application and providegeneral information regarding the technical field and provide assays andother details discussed in the application. The following references areincorporated herein by reference in their entirety.

-   [1] Prymula et al., Lancet, 367:740-48 (2006).-   [2] Vesikari et al., PIDJ, 28(4):S66-76 (2009).-   [3] Dagan et al. Infection & Immunity, 5383-91 (2004).-   [4] Juergens et al., Clinical and Vaccine Immunology,    21(9):1277-1281 (2014).-   [5] Andrews et al., Lancet, 14:839-846 (2014).-   [6] Nurkka et al., Vaccine, 20:194-201 (2001).-   [7] Levin and Stone, J. Immunology, 67:235-242 (1951).-   [8] W.H.O. Manual for the Production and Control of Vaccines:    Tetanus Toxoid, 1977 (BLG/UNDP/77.2 Rev.I)-   [9] Didierlaurent et al., J. Immunol., 183: 6186-6197 (2009).

1. A mixed carrier, multivalent pneumococcal conjugate composition,comprising 16 different pneumococcal capsular polysaccharide-proteinconjugates, wherein each pneumococcal capsular polysaccharide-proteinconjugate comprises a protein carrier conjugated to a capsularpolysaccharide from a different serotype of Streptococcus pneumoniae,wherein the Streptococcus pneumoniae serotypes are 1, 3, 4, 5, 6A, 6B,7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and 33F, wherein the proteincarrier is CRM₁₉₇ or tetanus toxoid, wherein two of the capsularpolysaccharides are conjugated to tetanus toxoid and the remainingcapsular polysaccharides are conjugated to CRM₁₉₇, and wherein the twocapsular polysaccharides that are conjugated to tetanus toxoid areselected from the group consisting of serotypes 1, 3, and
 5. 2. Themixed carrier, multivalent pneumococcal conjugate composition of claim1, wherein the capsular polysaccharides from serotypes 1 and 5 areconjugated to tetanus toxoid, and the capsular polysaccharides fromserotypes 3, 4, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and33F are conjugated to CRM₁₉₇.
 3. The mixed carrier, multivalentpneumococcal conjugate composition of claim 1, wherein the capsularpolysaccharides from serotypes 1 and 3 are conjugated to tetanus toxoid,and the capsular polysaccharides from serotypes 4, 5, 6A, 6B, 7F, 9V,12F, 14, 18C, 19A, 19F, 22F, 23F, and 33F are conjugated to CRM₁₉₇. 4.The mixed carrier, multivalent pneumococcal conjugate composition ofclaim 1, wherein the capsular polysaccharides from serotypes 3 and 5 areconjugated to tetanus toxoid, and the capsular polysaccharides fromserotypes 1, 4, 6A, 6B, 7F, 9V, 12F, 14, 18C, 19A, 19F, 22F, 23F, and33F are conjugated to CRM₁₉₇.
 5. The mixed carrier, multivalentpneumococcal conjugate composition of claim 1, further comprising anadjuvant.
 6. The mixed carrier, multivalent pneumococcal conjugatecomposition of claim 5, wherein the adjuvant is an aluminum-basedadjuvant.
 7. The mixed carrier, multivalent pneumococcal conjugatecomposition of claim 6, wherein the adjuvant is selected from the groupconsisting of aluminum phosphate, aluminum sulfate, and aluminumhydroxide.
 8. The mixed carrier, multivalent pneumococcal conjugatecomposition of claim 7, wherein the adjuvant is aluminum phosphate. 9.(canceled)
 10. A vaccine comprising the mixed carrier, multivalentpneumococcal conjugate composition of claim 1 and a pharmaceuticallyacceptable excipient.
 11. A method for prophylaxis of Streptococcuspneumoniae infection or disease in a subject, the method comprisingadministering a prophylactically effective amount of the mixed carrier,multivalent pneumococcal conjugate composition of claim 1 to thesubject.
 12. The method of claim 11, wherein the subject is a human whois at least 50 years old and the disease is pneumonia or invasivepneumococcal disease (IPD).
 13. The method of claim 11, wherein thesubject is a human who is at least 6 weeks old and the disease ispneumonia, invasive pneumococcal disease (IPD), or acute otitis media(AOM).
 14. The method of claim 13, wherein the subject is 6 weeks to 5years of age, 2 to 15 months of age, or 6 to 17 years of age.
 15. Themethod of claim 11, wherein the subject is a human.
 16. The method ofclaim 11, wherein the mixed carrier, multivalent pneumococcal conjugatecomposition or the vaccine is administered by intramuscular injection.17. The method of claim 11, wherein the mixed carrier, multivalentpneumococcal conjugate composition or the vaccine is administered aspart of an immunization series.